A phase I clinical trial of replication-competent Ad-HIV vaccines is soon to be initiated in the NIH clinical center in collaboration with NIAID and Dr. Mark Connors as Principal Investigator. The study will evaluate the safety and immunogenicity of Ad4-HIVenv and Ad4-HIVmosaic gag vaccines formulated as enteric coated capsules for oral administration and as a liquid for tonsillar application. A current clinical trial is on-going in which an Ad4-flu vaccine is being tested by the tonsillar route. This study will determine an appropriate dose for the Ad4 vector and will enable jump-starting of the Ad4-HIV vaccine studies. New Ad-recombinants are under development both for future clinical use and for pre-clinical studies in the rhesus macaque model. These include recombinants containing novel envelope inserts intended to generate broad, potent neutralizing antibodies. This work includes assessment of immunogenicity of the newly constructed recombinant vaccines in appropriate animal models. In addition, Ad vectors with deletion of genes not essential for replication of the virus have been developed, and will be used in future preclinical vaccine design. Previously, the STEP trial, a phase III clinical trial of a non-replicating Ad5-HIV vaccine showed lack of protective efficacy in people together with a suggestion that infection might have been enhanced in individuals with pre-existing immunity to Ad5. This trial was modeled in SIV rhesus macaques, and the results were recapitulated in showing no efficacy. However, enhancement of SIV infection following a penile challenge was not confirmed. On-going studies will further evaluate this issue. Overall, our project is focused on moving the replication-competent Ad-recombinant vaccine approach into phase I clinical trials. This is a novel approach, which has shown superior immunogenicity in non-human primate studies in comparison to non-replicating Ad-recombinant vaccines.